Induction of endothelial monolayer permeability by phosphatidate

Released into the vasculature from disrupted cells or transported to the su rface of adjacent effecters, phosphatidate and related lipids may potentiat e endothelial cell activation. However, the effect of these lipids on endot helial monolayer barrier integrity has not been reported. The present study documents the induction of endothelial monolayer permeability by phosphati date. Both long (di-C18:1) and medium (di-C10; di-C8) chain length phosphat idates increased permeability of bovine pulmonary artery endothelial cell m onolayers assessed using a well characterized assay system in vitro. Barrie r disruption effected by dioctanoyl (di-C8) phosphatidate was markedly pote ntiated by the addition of propranolol, an inhibitor of endothelial cell "e cto"-phosphatidate phosphohydrolase (PAP), a lipid phosphate phosphohydrola se (LPP) that efficiently hydrolyzes extracellular substrate. Disruption of barrier function by phosphatidate did not result from its non-specific det ergent characteristics, since a non-hydrolyzable but biologically inactive phosphonate analog of dioctanoyl phosphatidate, which retains the detergent characteristics of phosphatidate, did not induce permeability changes. Fur thermore, neither diacylglycerol nor lyse-PA effected significant increases in monolayer permeability, indicating the observed response was due to pho sphatidate rather than one of its metabolites. Phosphatidate-induced permea bility was attenuated by preincubation of endothelial cells with the tyrosi ne kinase inhibitor, herbimycin A(10 mu g/ml), and enhanced by the tyrosine phosphatase inhibitor, vanadate (100 mu M), implicating a role for activat ion of intracellular tyrosine kinases in the response. In addition, phospha tidate increased the levels of intracellular free Ca2+ in endothelial cells and ligated specific binding sites on endothelial cell plasma membranes, c onsistent with the presence of a phosphatidate receptor. Since phosphatidat e generated within the plasma membrane of adherent effecters potentially in teracts with endothelial membranes, we evaluated the influence of phosphati date-enriched neutrophil plasma membranes on endothelial monolayer integrit y. The effects of ectopic phosphatidate on endothelial monolayer permeabili ty were mimicked by phosphatidate confined to neutrophil plasma membranes. We conclude that phosphatidate may be a physiologic modulator of endothelia l monolayer permeability that exerts its effects by activating a receptor-l inked, tyrosine kinase-dependent process which results in mobilization of i ntracellular stored Ca2+ and consequent metabolic activation. (C) 1999 Wile y-Liss, Inc.